Advances in the chemical synthesis of oligoribonucleotides have not kept pace with the many advances in techniques developed for the chemical synthesis of oligodeoxyribo-nucleotides. The synthesis of RNA was actually a much more difficult task than the synthesis of DNA. The intemucleotide bond in native RNA was far less stable than in the DNA series. The close proximity of a protected 2′-hydroxyl to the intemucleotide phosphate presents problems, both in terms of the formation of the intemucleotide linkage and in the removal of the 2′-protecting group once the oligoribonucleotide has been synthesized (See FIG. 1).
Only recently has there been a great demand for small synthetic RNA. The discoveries of the RNAi pathway and small RNAs, such as siRNA, miRNAs and ntcRNAs associated with the RNA interference pathway was primarily responsible for this increased demand. Most recent attempts at the chemical synthesis of oligoribonucleotides have followed the synthetic strategy for the chemical synthesis of oligodeoxyribonucleotides: the standard phosphoramidite approach [Matteucci, M. D., Caruthers, M. H. J Am. Chem. Soc. 1981, 103, 3186-3191]. Such methods proceed by the step-wise addition of protected ribonucleoside phosphoramidite monomers to a growing RNA chain connected to a solid phase support. However, efficient solid phase synthesis of oligoribonucleotides still poorly compared to the efficiency of oligodeoxyribonucleotides synthesis.
Until recently, the typical approach to RNA synthesis utilized monomers whereby the 5′-hydroxyl of the ribonucleoside was protected by the acid-labile dimethoxytrityl (DMT) protecting group. Various protecting groups have been placed on the 2′-hydroxyl to prevent isomerization and cleavage of the intemucleotide bond during the acid deprotection step. By using this as a starting point for RNA synthesis, researchers have focused on finding an ideal 2′-protecting group compatible with acid deprotection. Research directed toward the discovery of this ideal 2′-protecting group has taken two primary courses: the use of acid-stable 2′-protecting groups and the use of acid-labile 2′-protecting groups. The use of acid-stable 2′-protecting groups has been quite limiting from a chemical perspective, since there are not many options available when the base lability of RNA was considered. Acid-stable protecting groups are typically base-labile or nucleophile-labile (e.g., removed by a strong base or a strong nucleophile). General base-labile protecting groups are removed by elimination or fragmentation subsequent to proton abstraction by a strong base. An example of this type of protecting group was a propionitrile-containing protecting group, which was removed by beta-elimination to form acrylonitrile after a proton was abstracted from the methylene carbon adjacent to the nitrile group. It was difficult to use these types of protecting groups on the 2′-hydroxyl of RNA since subsequent proton abstraction from the ensuing 2′-hydroxyl results in cleavage of the internucleotide bond via formation of a 2′-3′ cyclic phosphate and destruction of the RNA.

This approach was therefore only viable if the pH conditions used for proton abstraction from the protecting group are below pH 11, the pH at which proton abstraction from the 2′-hydroxyl begins to give rapid cleavage of the internucleotide bond. The approach of using a general base-labile protecting group for the 2′-hydroxyl has been further stymied by the necessary use of weak bases during the oxidation and capping reactions that occur in the standard phosphoramidite oligonucleotide synthesis process.
Protecting groups that are removed by the weakly basic conditions below pH 11 (such that the 2′-hydroxyl was not appreciably deprotonated) are typically unstable to the conditions used for capping and oxidation. As a result, the approach of using general base-labile protecting groups for 2′-hydroxyl protection has rarely been pursued, and never enabled.
Alternatively, there have been many attempts at the use of nucleophile-labile protecting groups for the protection of the 2′-hydroxyl. The difficulty associated with the use of nucleophile-labile protecting groups was that most typical nucleophiles are governed by the Brønsted-type plot of nucleophilicity as a function of basicity: the stronger the nucleophilicity, the stronger the basicity. As a result, strong nucleophiles are usually also strong bases and therefore the use of strong nucleophiles for deprotection of the 2′-hydroxyl typically results in the destruction of the desired RNA product by a subsequent proton abstraction from the 2′-hydroxyl. The use of nucleophile-labile 2′-hydroxyl protecting groups for RNA synthesis has only been enabled by the use of fluoride ion, a silicon-specific nucleophile that was reactive with silanes and siloxanes at a wide variety of pH conditions.
The most popular of these acid-stable protecting groups seem to be the t-butyl-dimethylsilyl group known as TBDMS [Ogilvie et al., Can. J. Chem., Vol 57, pp. 2230-2238 (1979)]. Widely practiced in the research community, the use of TBDMS as 2′-protecting group, dominated the previously small market for chemical synthesis of RNA for a very long time [Usman et al. J. Am. Chem. Soc. 109 (1987) 7845], [Ogilvie et al. Proc. Natl. Acad. Sci. USA 85 (1988) 5764]. The oligoribonucleotide syntheses carried out therewith are, however, by no means satisfactory and typically produces poor quality RNA products.
Several publications have reported the migration of the alkylsilyl group under a variety of conditions [Scaringe et al, Nucleic Acids Res 18, (18) 1990 5433-5441; Hogrefe et al. Nucleic Acids Research, 1993, 21 (20), 4739-4741]. Also, the loss of the 2′-silyl group that occurs during the removal of exocyclic amine protecting groups has been widely described in the literature [Stawinski et. al. Nucleic Acids Res. 1988, 16 (19), 9285-9298]. Methods that use less stable exocyclic amine protecting groups such as phenoxyacetyl or methoxyacetyl were subsequently developed to circumvent this problem [Schulhof et al. Nucleic Acids Res. 1987 15(2) 397-416]. However, the synthesis of the 5′-O-dimethoxytrityl-2 ′-O-tert-butyldimethylsilyl-ribo-3′-O-(β-cyanoethyl, N-diisopropyl)phosphoramidite monomers was still challenging and costly due to the non-regioselective introduction of the 2′-silyl group and the added chemical requirements to prevent migration of the silyl group during the phosphoramidite production. It was also well known in the art that the coupling efficiency of these monomers was greatly decreased due to the steric hindrance of the 2′-TBDMS protecting group, thereby affecting the yield and purity of the full-length product, and also limiting the length of the oligoribonucleotide that can be achieved by this chemical synthesis.
The most recent acid-stable 2′-hydroxyl protection approach for RNA synthesis was developed by Pitsch et al. [U.S. Pat. No. 5,986,084] to try to circumvent the problems encountered with the previous 2′-silyl protecting groups. This approach also relies on the use of 2′-O-acetals groups further protected by an alkylsilane, which was removed by the silicon-specific nucleophile fluoride ion. Although somewhat less acid stable than TBDMS, it was used in combination with acid-labile 5′-protecting groups such as DMT or the 5′-9-phenylxanthen-9-yl (Pix) group shown below.

Because of the presence of the methylene group, this 2′-protecting group was less bulky than the TBDMS, allowing higher coupling efficiency. Since the protecting group was an acetal moiety, there was no significant problem of isomerization. The commercial protecting group typically used in this approach was the tri-isopropyloxymethyl derivative known by the abbreviation TOM. Although this protecting group scheme solves many of the problems encountered by the TBDMS chemistry, it suffers from other significant difficulties. The synthesis of the TOM-protected monomers was extremely difficult and low yielding. The protecting group itself requires a low yield multi-step synthesis prior to its placement on the nucleoside. The attachment to the nucleoside was performed through a nucleophillic displacement reaction by a 2′-3′ alkoxide generated from a dialkyl tin reagent that produces a mixture of non-regioselective products that have to be separated and isolated by chromatography. In the case of the guanosine nucleoside, the tin reagents can preferentially react with the heterobase rather than the 2′-, 3′-hydroxyl moieties. In many cases, the overall yield of desired products from these reactions can be significantly less than 10%, rendering the monomer synthons and subsequent RNA products very expensive to produce.
Alternatively, many researchers have pursued the use of acid-labile groups for the protection of the 2′-hydroxyl moiety. The classic acid-labile protecting group was the 2′-acetal moiety, which was initially developed by Reese [Reese, C. B., Org. Biomol. Chem. 2005, 3(21), 3851-68], such as tetrahydropyran (THP) or 4-methoxy-tetrahydropyran (MTHP), 1-(2-chloroethoxy)ethyl (Cee) [O. Sakatsume et al. Tetrahedron 47 (1991) 8717-8728], 1-(2-fluorophenyl)-4-methoxypiperidin-4-yl (Fpmp) [M. Vaman Rao et al., J. Chem. Soc. Perkin Trans., Paper 2:43-55 (1993), Daniel C. Capaldi et al., Nucleic Acids Research, 22(12):2209-2216 (1994)].
One of the advantages of acetal protecting groups compared with silyl ethers protecting groups was that they can be introduced regioselectively into the 2′ position through the use of the Markiewicz protecting group: tetraisopropyldisiloxane- 1,3-diyl. This protecting group, also known as TIPS, simultaneously blocks the 5′- and 3′-hydroxyls to allow complete regioselective upon introduction of the acetal group on to the 2′- hydroxyl [Markiewicz W.T., J. Chem Research (S) 1979 24-25)]. Another advantage was that the phosphoramidite coupling with 2′-acetal protected monomers was typically more efficient than with trialkyl silanes. The problems encountered when using the combination of 5′-O-DMT and 2′-O-acetals groups reside in the difficulty to find suitable 2′-O acetal groups that are both completely stable to the anhydrous acidic conditions used to remove the 5′O-DMT group and completely labile to the mild aqueous acid conditions used to remove this 2′-acetal protecting group, while not cleaving the intemucleotide bond of the RNA. The removal of acetals that are stable under DMT deprotection conditions typically requires prolonged exposure to acidic conditions that degrade the RNA. To inhibit the loss of the 2′ protecting group, the 5′-9-phenylxanthen-9-yl (Pix) group was applied, which was more labile than the DMT protecting group.
Even considering all of these innovations, the inability to find a viable combination of 2′-acetal and 5′-acid labile protecting groups that fits into the standard phosphoramidite synthesis cycle has resulted in these chemical schemes that were never effectively commercialized. Conversely, acetals used in combination with 5′ protecting groups such as leuvinyl and 9-fluorenylmethyloxycarbonyl (FMOC) that are deprotected under non-acidic conditions like hydrazinolysis have not met significant success. One of the overriding reasons that 2′-acetals have not achieved wide acceptance was that they tend to be too stable under the required acid deprotection conditions once the monomers are incorporated onto an oligonucleotide, due to the close proximity of the protected 2′-hydroxyl to the internucleotide phosphate. There was a significant change in the stability of the protecting group once the oligonucleotide was produced. Conditions that can effectively remove an acetal group from a protected nucleoside monomer tend to be ineffective to remove the same group from the oligonucleotide.
To address this issue, Dellinger et al. developed 2′-orthoester protecting groups whose labiality on the oligonucleotide was less affected by close proximity to the intemucleotide phosphate allowing effective removal under aqueous acid conditions that do not degrade the desired RNA product. The use of 2′-cyclic orthoesters was evaluated using a regioselective coupling procedure as well as a set of 5′-nucleophile labile carbonates [Marvin H. Caruthers, Tadeusz K. Wyrzkiewicz, and Douglas J. Dellinger. “Synthesis of Oligonucleotides and Oligonucleotide Analogs on Polymer Supports” In Innovation and Perspectives in Solid Phase Synthesis: Peptides, Proteins and Nucleic Acids (R. Epton, ed.) Mayflower Worldwide Limited, Birmingham, 39-44 (1994)]. Subsequently, Scaringe et. al. developed a set of 5′- and 2′-protecting groups that overcome the problems associated with use of 5′-DMT. This method uses a 5′-silyloxy protecting group [patents U.S. Pat. Nos. 5,889,136, 6,111,086, and 6,590,093] which require silicon-specific fluoride ion nucleophiles to be removed, in conjugation with the use of optimized 2′-orthoesters protecting groups (ACE). Although the coupling efficiency was greatly increased with the use of the ACE 2′-orthoester protecting group, and the final deprotection facile under pH conditions at which RNA was stable, the use of fluoride anions to deprotect the 5′-protecting groups prior each condensation cycle carries some disadvantages for routine synthesis of RNA and was even more problematic for large-scale synthesis of RNA. Because this chemistry requires atypical nucleoside protecting groups and custom synthesized monomers, namely on the 5′OH, it was difficult and time consuming to build RNA sequences that contain other commercially available phosphoramidite monomers, such as modified nucleotides, fluorescent labels, or anchors.
In order to incorporate a wide variety of alternative monomers and modifications using this chemistry, it was necessary to have each of them custom-synthesized with the appropriate 5′-silyloxy protecting group, thus significantly limiting the commercial applications for this chemistry. The ACE chemistry has the ability to produce very high quality RNA, but the reactions conditions are tricky and the synthesis not robust enough to routinely produce long sequences of RNAs. As a result, there was still clearly a need for the development of a chemical synthesis method for RNA that was simple and robust and produces high quality RNA products, while fitting into the standard phosphoramidite oligonucleotide synthesis approach. The commercial success of the ACE chemistry clearly illustrates the need to develop a RNA synthesis method that was founded upon mild and simple final deprotection conditions that will not affect the integrity of the final RNA product.
While protected, the RNA molecule has similar stability to the DNA molecule. Consequently, the final deprotection conditions to treat a synthetic RNA molecule are typically the same as the conditions to treat a synthetic DNA molecule prior to the removal of the 2′-hydroxyl protecting group. As a result, the current methods of RNA synthesis perform the final deprotection of the synthetic RNA in a 4-, 3-, or a 2-step fashion.                1. Deprotection of the protected phosphotriester, most commonly the cyanoethyl group (CNE), which was performed by brief exposure to ammonia (½ hr at room temperature) or in the case of the methyl group, by treatment with thiophenol for ½ hr at room temperature.        2. Cleavage of the oligoribonucleotides from the support performed under basic conditions, usually by exposure to ammonium hydroxide, anhydrous ammonia in an alcohol, methylamine, other alkyl amines, basic non-amine solutions such as potassium carbonate solutions, or non-nucleophillic bases such as 1,8-diazabicyclo[5.4.0]undec-7-ene (DBU) in organic solvents.        3. Deprotection of the nucleobases, which one most commonly protected on the exocyclic amine with protecting groups such as phenoxyacetyl (PAC), acetyl (Ac), isobutyryl (iBu) or benzoyl (Bz) and which are typically also removed under basic conditions. Most of the time, steps 2 and 3 are performed simultaneously.        4. Usually, the 2′-deprotection was performed post-cleavage of the oligonucleotide from the support. In the case of TBDMS and TOM chemistry, the oligoribonucleotide was exposed to fluoride anions to deblock the 2′ hydroxyl groups after cleavage of the oligoribonucleotide from the support. In the case of the ACE chemistry, the 2′-O-orthoester group can be removed with acidic conditions after cleavage of the oligoribonucleotide from the support, but also can be kept and deprotected during shipment to the customers or after shipment by the customers (this procedure allows keeping the oligoribonucleotide intact longer, since RNA was very sensitive to nuclease RNase degradation).Steps 1-3 can be performed simultaneously, when appropriate, making it a 2-step deprotection process, or step 1 can be performed independently, and steps 2-3 combined, making it a 3-step final deprotection process.        
The removal of the 2′-hydroxyl protecting group was problematic for both the 3- and 2-step processes. In the 3-step process, the phosphorus protecting group was typically removed first, while the oligoribonucleotide was still attached to a solid support. In the second step, the heterobase protecting groups are removed using a nucleophillic base like ammonia or methyl amine, also which usually result in the cleavage of the oligoribonucleotide from the support.
Finally, a fluoride ion-based solution under neutral, mildly acidic, or mildly basic conditions (TBDMS, TOM) [Pitsch, et. al. Helv. Chim. Acta, 2001, 84, 3773-3795] or a weak acidic solution was used to remove the ACE 2′-hydroxyl protecting group [Scaringe et al, Nucleic Acids Res 18, (18) 5433-5441 (1990); Scaringe et al, J. Am. Chem. Soc., 120, 11820-11821 (1998)]. This process requires that the 2′-hydroxyl protecting gropup was orthogonally stable to the deblock conditions utilized to remove the protecting group for the 3′- or 5′-hydroxyl functional during the chemical synthesis process, and stable to the conditions utilized for deprotection of the phosphorus protecting groups and the heterobase protecting groups. Most often it was seen that a loss of the 2′-protecting group occurs to some extent during one of these previous deblock or deprotection processes. The result was modification of the desired RNA strand or cleavage of the desired RNA product.
Modification and cleavage decreases the yield and quality of the desired RNA products and can often prevent synthesis and isolation of oligonucleotide sequences significantly longer than 15 or 20 nucleotides in length. In the case of the use of a fluoride ion solution for deprotection of the 2′-hydroxy group, removal of residual fluoride ions requires additional steps and can be quite difficult and time consuming.
In the 3-step process, removal of the phosphorus protecting groups was accomplished simultaneously with the removal of the heterobase protecting groups. This was usually accomplished using a nucleophillic base like ammonia or methylamine. Most often the phosphorus-protecting group was removed using a beta-elimination reaction such as the formation of acrylonitrile from a 3-hydroxypropionitrile ester. However, the use of this system for the protection of the intemucleotide phosphodiester linkage, followed by simultaneous deprotection during cleavage of the heterobase protecting groups, results in a number of notable side reactions that affect the yield and purity of the final product. The use of protecting groups that are susceptible to cleavage by proton abstraction followed by beta-elimination generally decreases the reactivity of the active phosphorus intermediate due to their electron withdrawing nature, and this effect lowers the per-cycle coupling efficiency. In addition, the elimination products such as acrylonitrile are reactive toward the heterobases and often form base adducts that result in undesired modifications.